Substrate phosphorylation specific activity of EGF receptor is increased 500-fold (from a turnover number of 2/min to 1000/min at 30C) in an allosteric process involving bimolecular interactions between receptor molecules and an over 100-fold increase in ATP Km. This is reminiscent of the process underlying the unisite and multisite catalytic activities of F1 ATPase. Vesicles with only very high affinity EGF binding (KD=0.1nM) were isolated free of membranes with lower affinity (KD=2-5nM) EGF binding. Receptors in these vesicles are activated for multisite high specific activity substrate phosphorylation activity. We postulate that EGF activates a unimolecular receptor self-phosphorylation process that initiates receptor internalization in a specialized class of vesicles with high receptor density to support the multisite mode of substrate phosphorylation. The experiments proposed will: 1. a. Define the multisite mechanism further in terms of rate determining processes requiring high receptor density, increased ATP Km, and overcoming of strong ADP end product inhibition; b. Characterize inhibitory properties of polypeptides which may act by disrupting allosteric receptor interactions required for multisite substrate phosphorylation activity; and c. Reconstruct in phospholipid vesicles and characterize the system catalyzing multisite substrate phosphorylation; 2. a. Identify the pivotal reaction in which EGF is critical for activating multisite substrate phosphorylation; b. Define how protein kinase C disrupts this process; 3. Refine procedures for purifying vesicles with very high affinity EGF receptors to characterize their multisite substrate phosphorylation properties; and 4. Attempt to define components other than receptor which are obligatory for specific receptor internalization leading to activation of multisite substrate phosphorylation activity. In an unrelated study, EGF induced tyrosine phosphorylation of human glucocorticoid receptor in cultured cells; this was correlated with decreased glucocorticoid binding. Purified glucocorticoid receptor will be phosphorylated by purified EGF receptor to test for decreased ligand binding activity.